Construction & expression in E. coli of novel single-chain antibody fragments against the herbicide atrazine
The objective of this research was to determine the potential of two anti-atrazine single-chain antibodies (scAbs) to detect very dilute atrazine concentration, specifically in the nanomolar and sub-nanomolar ranges. Analysis of environmental pesticide contamination is routinely done by high performance liquid chromatography (HPLC), gas chromatography (GC) and mass spectrometry (MS). However, since the mid eighties there have been an increasing number of reports describing the use immunoassays to detect environmental pollutants. Although immunoanalysis using whole antibodies has been shown to be almost as effective as GC/MS (Thurman et al., 1990) the technology is still not widely used for pesticide analysis. This thesis will describe the increased sensitivity of single-chain antibody fragments, compared to whole antibodies, to detect low concentrations of atrazine and related triazines. The effect of monomeric and dimeric scAb conformations on the ability of enzyme linked immunosorbent assays (ELISAs) to detect atrazine and related triazines is investigated. The thermal and chemical stability of the scAbs, and a modified scAb containing an interchain disulphide bond, are compared with the parent monoclonal antibodies (MAbs) to determine their suitability in an environmental ELISA assay system. The expressions in Escherichia coli (E. coli) of two different anti-atrazine scAbs and related antibody fragments are studied, and an optimised expression protocol for these antibody fragments obtained. Three potentially toxic amino acid residues identified in one of the variable heavy (VH) domains (Knappik & Pluckthun, 1995) are mutated to less toxic amino acids, and their effects on antibody fragment expression observed.